Hemoglobin is a heterotetramer composed of two alpha and two beta-globin chains. The proteins are encoded by two different gene loci. Many human genetic diseases are associated with mutations in the human beta-globin gene locus and lead to mild or severe forms of anemia. Treatment options for severe cases are limited and often accompanied by deleterious side effects. The most common mutations that cause hemoglobinopathies reduce expression of the adult beta-globin gene. It is anticipated that knowledge about how the globin genes are regulated during differentiation and development will lead to new forms of treatement involving gene and stem cell therapies. The human beta-globin gene locus consists of five genes that are sequentially expressed during development exclusively in erythroid cells. High-level expression of these genes is mediated by a locus control region, a powerful and complex DMAregulatory element located far upstream of the genes. The locus control region is remarkable in that it is able to confer position- independent and high-level expression to globin genes in transgenic assays This activity is important in gene therapy experiments, which are aimed at expressing physiological levels of a therapeutic globin gene in erythroid cells. Recent evidence suggests that the locus control region, which consists of several core regions harboring many transcription factor binding sites, recruits activities that are required for establishing accessible chromatin domains in the beta-globin locus. Moreover, several recent reports demonstrate that RNA polymerase II is recruited to locus control region core elements. We hypothesize that the LCR represents the primary attachment site for recruitment of transcription complexes, which are delivered to the globin genes in a developmental stage specific manner. We will use biochemical, molecular cell biological, and genetic experiments to test this model. In addition, we will investigate the role of helix-loop-helix proteins USF and TFII-I in beta-globin gene regulation. Our data suggest that these proteins antagonistically regulate expression of the adult beta-globin gene and may participate in the stage-specific expression of the globin genes. We will generate transgenic mice expressing dominant negative mutants of these proteins and analyze the consequence of expression of these proteins on globin gene regulation during mouse development.